OP0097 PROOF-OF-CONCEPT OF FIBROBLAST-TARGETED PHOTODYNAMIC THERAPY IN HUMAN 3D SCLERODERMA SKIN MODEL AND BLEOMYCIN-INDUCED SKIN FIBROSIS IN MICE
Authors: D. Dorst, A. van Caam, E. Vitters, B. Walgreen, M. Helsen, C. Klein, S. Gudi, T. Wubs, J. Kumari, P. Błyszczuk, M. Vonk, P. van der Kraan, M. Koenders
Journal: Annals of the Rheumatic Diseases (2022)

Background Systemic sclerosis (SSc) is a rare, severe auto-immune disease characterized by inflammation, vasculopathy and fibrosis. Activated (myo)fibroblasts are crucial drivers of fibrosis. By exploiting their expression of fibroblast activation protein (FAP) to perform targeted photodynamic therapy (tPDT), we can locoregionally deplete these pathogenic cells. Objectives We explored the use of FAP-tPDT to selectively target primary skin fibroblasts from SSc patients, both in 2D and 3D cultures, as well as in biopsies from fibrotic skin lesions in the murine bleomycin-induced skin fibrosis model. Methods The FAP targeting monoclonal antibody (clone 28H1) was conjugated with the photosensitizer IRDye700DX. Primary skin fibroblasts were obtained from lesional skin biopsies of SSc patients via spontaneous out-growth and subsequently cultured on plastic or collagen type I. For 2D FAP-tPDT, cells were incubated in buffer with or without the antibody-photosensitizer (Ab-PS) construct, washed after 4 h and exposed to λ = 689 nm light (50 J/cm 2 at 280 mW/cm 2 ). Cell viability was measured using CellTiter Glo. For 3D FAP-tPDT, cells were seeded in collagen plugs and underwent the same treatment procedure. Contraction of the plugs was followed over time to determine myofibroblast activity. Skin fibrosis was induced in mice by 3x/week injections of 1.5 IU bleomycin intradermally on the back of the mouse. After 5 weeks the mice were sacrificed and biopsies were taken from affected and not affected skin. These were incubated with or without the FAP targeting Ab-PS construct or a control Ab-PS. After washing, the biopsies were exposed to light, incubated for 1h and subsequently formalin fixed and paraffin embedded. Sections were immuno stained for the presence of cleaved caspase-3. Results FAP-tPDT resulted in antibody-dose dependent cytotoxicity in primary skin fibroblasts upon light exposure. Cells not exposed to light or incubated with a control Ab-PS construct did not show this response. FAP-tPDT fully prevented contraction of collagen plugs seeded with primary SSc fibroblasts. Even incubation with a very low dose of antibody (0.4 nM) inhibited contraction in 2 out of 3 donors. In fibrotic skin biopsies from mice with bleomycin-induced skin fibrosis, an upregulation of apoptosis, as evidenced by increased caspase-3 staining (Figure 1), was observed in response to FAP-tPDT. The same treatment on biopsies of control skin did not increase caspase-3 staining, nor did incubation with a control Ab-PS construct or light alone. Figure 1. Cleaved caspase-3 staining of biopsies from the skin of mice with bleomycin-induced dermal fibrosis upon treatment with FAP-tPDT. The red arrows indicate the presence of positive fibroblasts. Conclusion Here we have shown, for the first time, the potential of FAP-tPDT for the selective targeting of pathogenic fibroblasts and treatment of fibrosis in SSc skin both in relevant patient-derived culture models as well as an in vivo model of fibrosis. Disclosure of Interests Daphne Dorst: None declared, Arjan van Caam: None declared, Elly Vitters: None declared, Birgitte Walgreen: None declared, Monique Helsen: None declared, Christian Klein Employee of: CK is an employee of Roche pharmaceutics, Shreya Gudi: None declared, Tirza Wubs: None declared, Jyoti Kumari: None declared, Przemysław Błyszczuk: None declared, Madelon Vonk: None declared, Peter van der Kraan: None declared, Marije Koenders: None declared.